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Thesis Sci 2006 Karreman Rj (1).Pdf The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town THE LOCALIZATION, FUNCTION AND APPLICATIONS OF THE STRESS RESPONSE PROTEIN HSP12P IN THE YEAST SACCHAROMYCES CEREVISIAE. by Robert Jan Karreman Town Cape A thesis submittedof for the degree of Doctor of Philosophy in the Department of Molecular and Cell Biology, Faculty of Science University of Cape Town, South Africa. University March 2006 PREFACE The work presented in this thesis was perfonned under the supervision of Associate Professors George G. Lindsey and WolfF. Brandt at the Department of Molecular and Cell Biology, University of Cape Town, South Africa. I hereby declare that this thesis, submitted for the degree of Doctor of Philosophy in Molecular and Cell Biology at the University of Cape Town, is the result of my own investigation, except where the work of others is acknowledged. Robert Jan Karreman University of Cape Town March 2006 University of Cape Town ACKNOWLEDGEMENTS [ would like to thank the following people and/or organizations: My supervisor, George Lindsey, for being an excellent and understanding mentor. My parents, "Rube" Karreman and Louise Karreman, for all their dedication, love and support throughout my studies with special thanks to my dad for all the time on the MS. My fiance, Carmin Trader, the love of my life, for giving me emotional support and many hugs and laughs. My in-laws to-be, Denise and Robert Trader, for their love and support. Dr. Florian Bauer, for the YCP-GFP2 plasmid. Dr. Peter Meacock, for yeast strains. Assoc. Prof. Hugh Patterton and members of his laboratory, for the pYES2 plasmid and assorted molecular biology reagents. Dr. Suhail Rafudeen, for help with Realtime PCR. Town Prof. Pieter van Wyk, for help with confocal microscopy. Dr. Fabien Gaboriaud and his wife, for their hospitality and help in Atomic Force Microscopy conducted in Nancy, France. Cape Etienne Dague, Fabienne Quiles and Jeromeof Duval for their assistance with Atomic Force Microscopy, Raman spectroscopy and electrophoretic mobility assays respectively. Andrew Kessler, for being my friend since 1st year and helping me get through the tougher times, with many laughs along the way. John Moore, for his witty comments and infectious humour. Pat Thompson, for helpingUniversity me find things in the lab and for advice. Di James and Pei-yin Ma for DNA sequencing and oligonucleotide synthesis respectively. All the people in Lab 402 for their great humour and for providing a pleasant atmosphere to work 1D. The National Research Foundation, for funding throughout the project and for bursaries and a Scarce Skills Scholarship. The University of Cape Town, for funding a part of my studies and for Research Associateship awards. The Benfara Trust for funding a part of my studies. I TABLE OF CONTENTS ABSTRACT II ABBREVIATIONS IV LIST OF FIGURES & TABLES VIII Town CHAPTER 1 GENERAL INTRODUCTION 1 Cape of THE LOCALIZATION AND CHAPTER 2 24 APPLICATIONS OF HSP12P CHAPTER 3 THE FUNCTION OF HSP12P 54 University CHAPTER 4 GENERAL DISCUSSION 114 REFERENCES 117 n THE LOCALIZATION, FUNCTION AND APPLICATIONS OF THE STRESS RESPONSE PROTEIN HSP12P IN THE YEAST SACCHAROMYCES CEREVISIAE. by Robert Jan Karreman March 2006 Department of Molecular and Cell Biology, Faculty of Science University of Cape Town, Cape Town, South Africa ABSTRACT Since 1990, the yeast Saccharomyces cerevisiae small heat shockTown protein Hsp 12p, has continuously appeared in data associated with stress responses in this organism. Hsp 12p is expressed abundantly in response to a large variety of di fferent stresses, but for many years has eluded researchers as to its function, primarily because the viability of yeast strains lacking HSP 12 are unaffectedCape by osmotic stress and heat shock. Subsequent studies indicated that Hspof 12p played a role in the adaptation of the cell wall of Saccharomyces cerevisiae to conditions of stress. However, the exact in vivo localization, speci fic function and mediation of function of Hsp 12p had yet to be elucidated. The localization ofUniversity Hsp 12p was determined by fusion to the green fluorescent reporter protein, Gfp2p and a combination of epifluorescent microscopy and confocal imagery. Chemical extraction revealed that Hsp 12p was present in the cell wall while fluorescent imagery was not conclusive. This fluorescent Hsp 12p construct was later employed in a novel application to sense the stress status of yeast, which bears future promise for use in an industrial setting. The localization of Hsp 12p in the cell wall and its massive induction upon stress prompted the investigation into stress factors affecting cell wall status. Yeast cells were exposed to the cell wall damaging agent, Congo Red (CR) and the viability, morphology and dimorphic (invasive) growth of yeast cells lacking HSP 12 III investigated. Hsp 12p was found to play an essential role in the viability of yeast cells exposed to CR and in addition, was required for invasive growth in the presence of CR. Since these phenotypes were very similar to those observed in yeast cells lacking another class of proteins, the PIR (proteins with inverted repeats) proteins, levels of these proteins were compared in the wildtype and HSP J2-deficient yeast strains, but no differences were observed. The method by which Hsp 12p mediated its function was investigated by in vitro studies using chitin and CR, which revealed that Hsp 12p bound this polymer and prevented CR binding. The role ofHsp12p in invasive growth was detemlined by assessing the regulation of proteins involved in this process (flocculins) using Realtime PCR. These studies revealed that Hsp 12p did not affect flocculin regulation and thus acted through a different mechanism. Throughout these experiments, the HSP 12-deficient strain showed an increase in flocculation ability. The nature of this effect was detenninedTown by hydrophobicity studies using octane partitioning, but these studies indicated that Hsp 12p did not affect cell hydrophobicity and thus flocculation occurred through a different mechanism. Cape These findings implied that Hsp 12p actedof directly on the yeast cell wall and did not affect other proteins in this locus. Thus the direct effects of Hsp 12p on the cell wall were investigated. These studies included the use of agarose as a model system to simulate the yeast cell wall, which showed that Hsp 12p increased cell wall flexibility. Further studies using atomic force microscopy confinned that Hsp 12p increased the flexibility of the cellUniversity walls of live yeast cells. Studies of cell electrophoretic mobility revealed that cells lacking Hsp12p had stiffer cell walls. Finally, cell wall chemistry was assayed with infrared spectroscopy. Decreased phosphorylation of peptidomannan residues and a lower ~-1 ,3-glucan content was observed in Hsp 12p­ deficient cells, possibly due to differential regulation of calcium ion flux. The decrease in cell wall polysaccharides was proposed to result in a compensatory increase in the amount of chitin in the cell wall. This increase correlated with the flocculation and CR-sensitive phenotype displayed by cells lacking Hsp 12p, since chitin levels must be controlled for optimal cell separation and chitin is the target for CR binding. IV ABBREVIATIONS alpha beta delta delta, indicates gene mutations lambda rho sIgma micro, indicates 10-6 degrees Celcius microgram (10-6 g) microliter (10-3 ml) 3-D three dimensional A adenine AFM atomic force microscopy A. nidl/lans Aspergillus nidulans Town ARE API response element ATP adenosine triphosphate ATR attenuated total reflectance BCIP 5-bromo-4-chloro-3-indolylCape phosphate BLAST Basic Local Alignment Search Tool bp base pair(s) of BSA bovine serum albumin C cytosine 2 Ca + calcium ion C. alhicalls Candida alhicans cAMP cyclic adenosine monophosphate cDNA complementary DNA CH3CN Universityacetonitrile CHCA a-cyano-4-hydroxy cinnamic acid CIS cell integrity stress cm centimeter CR Congo Red CSM complete synthetic growth medium cw cell wall l Da dalton (g.mor ) DNA deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate DTT dithiothreitol E. coli Escherichia coli EDTA ethylenediaminetetra-acetic acid v ELISA enzyme-linked immunosorbance assay EtBr ethidium bromide FTIR Fourier Transform Infrared G guamne g gram 2 g standard gravitational acceleration (9.8 m.s- ) GFP green fluorescent protein GSR general stress response h hours HIS histidine HOG high osmolarity glycerol Hsp heat shock protein HSE heat shock element HSR heat shock response IPTG isopropyl-~-D-thiogalactopyranoside IR infrared Town k spring constant KCl potassium chloride kb kilobase pairs (103 bp) KBr potassium bromide kDa kilo dalton (103 Da) Cape kN kilonewton (103 N) KN03 potassi um nitrate of KO hsp12L1 URA3 S. cerevisiae strain liter LB Luria-Bertani growth medium LEA late embryogenesis-abundant m meter M Universitymolar (mol.rl) m/z mass per charge mA milliampere (10-3 A) MALDI-TOF matrix assisted laser desorption/ionisation time of flight MAP mitogen-activated protein MCS multiple cloning site mg milligram (10-3 g) MgCb magnesium chloride mm minutes ml milliliter (10-3 I) mm millimeter (10-3 m) mM millimolar (10-3 M) MPa megapascal (103 Pa) mRNA
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